PROJECT SUMMARY/ABSTRACT Epigenetic mechanisms play a critical role in controlling gene expression during normal development and are often altered in disease states, particularly in cancer. MLL-rearranged (MLL-r) leukemias account for 5-10% of human acute leukemia and is associated with poor prognosis. The unmet clinical needs and the lack of effective targeted therapy to the MLL-r leukemias emphasize the need for novel regimens. Recent cancer epigenetics studies discovered a central role for the histone H3 lysine 79 (H3K79) methyltransferase DOT1L in MLL-r leukemogenesis. Important clinical responses have been noted with DOT1L inhibitor treatment as a single agent, however, it is expected that combination treatments will be necessary. Our preliminary studies proposed in the parent award R01 CA236626 based on a high-density CRISPR genetic screen of DOT1L have identified the potential of ?saturation CRISPR protein scan? in de novo functional domain discovery. The objective of this Revision Application is to establish a computational pipeline to facilitate the data analyses of the saturation CRISPR protein scan. Our central hypothesis is that saturation CRISPR protein scan data harbor clinically impactful information that can be further explored through adaptation of the integrative informatics tools from the ITCR Program. We will (Aim 1) integrate the saturation CRISPR protein scan with cBioPortal (an integrative clinical genomic database; funded by ITCR) to identify clinically relevant alleles, and (Aim 2) streamline the saturation CRISPR protein scan with DINC 2.0 (a parallelized meta- docking method for the incremental docking of large ligands; funded by ITCR) to identify lead epigenetic ligands to the CRISPR defined hotspots. This study is innovative because (1) it introduces a novel concept of using saturation CRISPR genetic screens for epigenetic ligand discovery, and (2) it establishes an analysis pipeline for a novel field that bridges the functional genomics, protein domain identification, and therapeutics discovery. The impact of this research will be of significance because (1) it immediately provides novel therapeutic opportunities against the difficult-to-treat MLL-r leukemias, and (2) the computational pipeline established in this proposal and streamlined with the ITCR supported integrative informatics tools will help identify novel functional elements for future pharmaceutical targeting in multiple types of cancers.